U.S. patent application number 09/965116 was filed with the patent office on 2002-09-26 for modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes.
Invention is credited to Agrawal, Sudhir, Kandimalla, Ekambar R., Yu, Dong, Zhao, Qiuyan.
Application Number | 20020137714 09/965116 |
Document ID | / |
Family ID | 27398718 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137714 |
Kind Code |
A1 |
Kandimalla, Ekambar R. ; et
al. |
September 26, 2002 |
Modulation of immunostimulatory activity of immunostimulatory
oligonucleotide analogs by positional chemical changes
Abstract
The invention relates to the therapeutic use of oligonucleotides
or oligonucleotide analogs as immunostimulatory agents in
immunotherapy applications. The invention provides methods for
enhancing the immune response caused by immunostimulatory
oligonucleotide compounds.
Inventors: |
Kandimalla, Ekambar R.;
(Southboro, MA) ; Zhao, Qiuyan; (Southboro,
MA) ; Yu, Dong; (Westboro, MA) ; Agrawal,
Sudhir; (Shrewsbury, MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
27398718 |
Appl. No.: |
09/965116 |
Filed: |
September 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09965116 |
Sep 26, 2001 |
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09712898 |
Nov 15, 2000 |
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60235453 |
Sep 26, 2000 |
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60235452 |
Sep 26, 2000 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
C12N 2310/334 20130101;
C12N 2310/33 20130101; C07H 21/00 20130101; C12N 2310/32 20130101;
C12N 2310/321 20130101; A61P 31/12 20180101; A61P 31/04 20180101;
A61P 37/04 20180101; A61P 11/00 20180101; C12N 2310/336 20130101;
A61K 31/7088 20130101; A61P 33/00 20180101; A61P 37/08 20180101;
A61P 35/00 20180101; C12N 2310/3125 20130101; A61K 31/7115
20130101; C12N 15/117 20130101; C12N 2310/18 20130101; A61K
2039/55561 20130101; C12N 2310/317 20130101; C12N 2310/318
20130101; C12N 2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
514/44 ;
536/23.1 |
International
Class: |
C07H 021/04; A61K
048/00 |
Claims
What is claimed is:
1. An immunostimulatory oligonucleotide compound, comprising an
immunostimulatory dinucleotide of formula 5'-pyrimidine-purine-3',
wherein pyrimidine is a non-natural pyrimidine nucleoside and
purine is a natural or non-natural purine nucleoside.
2. An immunostimulatory oligonucleotide compound, comprising an
immunostimulatory dinucleotide of formula C*pG, wherein C* is a
cytidine analog, G is guanosine, 2'-deoxyguanosine, or a guanosine
analog, and p is an internucleotide linkage selected from the group
consisting of phosphodiester, phosphorothioate, and
phosphorodithioate.
3. The immunostimulatory oligonucleotide compound of claim 1,
wherein the non-natural pyrimidine nucleoside has the formula (I):
2wherein D is a hydrogen bond donor, D' is selected from the group
consisting of hydrogen, hydrogen bond donor, hydrogen bond
acceptor, hydrophilic group, hydrophobic group, electron
withdrawing group and electron donating group, A is a hydrogen bond
acceptor or a hydrophilic group, X is carbon or nitrogen, and S is
a pentose or hexose sugar ring, provided that the pyrimidine
nucleoside of formula (I) is not cytidine or deoxycytidine.
4. The immunostimulatory oligonucleotide compound of claim 3,
wherein the non-natural pyrimidine nucleoside includes a
non-naturally occurring pyrimidine base.
5. The immunostimulatory oligonucleotide compound of claim 4,
wherein the non-naturally occurring pyrimidine base is selected
from the group consisting of 5-hydroxycytosine,
5-hydroxymethylcytosine, N4-alkylcytosine, and 4-thiouracil.
6. The immunostimulatory oligonucleotide compound of claim 4,
wherein the non-naturally occurring pyrimidine base is selected
from the group consisting of 5-hydroxycytosine and
N4-ethylcytosine.
7. The immunostimulatory oligonucleotide compound of claim 4,
wherein the non-natural pyrimidine nucleoside of formula (I)
comprises a non-naturally occurring sugar moiety.
8. The immunostimulatory oligonucleotide compound of claim 7,
wherein the non-naturally occurring sugar moiety is arabinose.
9. An immunostimulatory oligonucleotide compound comprising an
immunostimulatory domain of formula (II): 5'-X1-X2-Y-Z-X3-X4-3'
(II) wherein Y is cytidine, 2'-deoxycytidine, or a non-natural
pyrimidine nucleoside; Z is guanosine, 2'-deoxyguanosine, or a
non-natural purine nucleoside; X1 is a naturally occurring
nucleoside or an immunostimulatory moiety selected from the group
consisting of C3-alkyl linker, 2-aminobutyl-1,3-propanediol linker,
and .beta.-L-deoxynucleoside- ; X2 is a naturally occurring
nucleoside or an immunostimulatory moiety that is an amino linker;
X3 is a naturally occurring nucleoside an immunostimulatory moiety
that is a nucleoside methylphosphonate; X4 is a naturally occurring
nucleoside an immunostimulatory moiety selected from the group
consisting of nucleoside methylphosphonate and
2'-O-methyl-ribonucleoside; provided that at least one of X1, X2,
X3, and X4 is an immunostimulatory moiety.
10. The immunostimulatory oligonucleotide compound of claim 9,
wherein Y is a non-natural pyrimidine nucleoside.
11. The immunostimulatory oligonucleotide compound of claim 10,
wherein Y has the formula (I): 3wherein D is a hydrogen bond donor,
D' is selected from the group consisting of hydrogen, hydrogen bond
donor, hydrogen bond acceptor, hydrophilic group, hydrophobic
group, electron withdrawing group and electron donating group, A is
a hydrogen bond acceptor or a hydrophilic group, X is carbon or
nitrogen, and S is a pentose or hexose sugar ring, provided that Y
is not cytidine or deoxycytidine.
12. An immunostimulatory oligonucleotide compound comprising a
sequence of formula (III): 5'-Um . . . U1-X1-X2-Y-Z-X3-X4-D1 . . .
Dm-3' (III) wherein: Y is a non-natural pyrimidine nucleoside; Z is
guanosine, 2'-deoxy-guanosine or a non-natural purine nucleoside;
each X independently is a naturally occurring nucleoside or an
immunostimulatory moiety; wherein Um-U1 represents an upstream
potentiation domain, where each U independently is a naturally
occurring nucleoside or an immunostimulatory moiety; wherein D1-Dm
represents a downstream potentiation domain, where each D
independently is a naturally occurring nucleoside or an
immunostimulatory moiety; and m, at each occurrence, represents a
number from 0 to 30.
13. The immunostimulatory oligonucleotide compound of claim 12,
wherein at least one X, U, or D is an immunostimulatory moiety.
14. The immunostimulatory oligonucleotide compound of claim 13,
wherein: X1 is a naturally occurring nucleoside or an
immunostimulatory moiety selected from the group consisting of
C3-alkyl linker, 2-aminobutyl-1,3-propanediol linker, and
.beta.-L-deoxynucleoside; X2 is a naturally occurring nucleoside or
an immunostimulatory moiety that is an amino linker; X3 is a
naturally occurring nucleoside an immunostimulatory moiety that is
a nucleoside methylphosphonate; X4 is a naturally occurring
nucleoside an immunostimulatory moiety selected from the group
consisting of nucleoside methylphosphonate and
2'-O-methyl-ribonucleoside; U1 is a naturally occurring nucleoside
an immunostimulatory moiety selected from the group consisting of
1',2'-dideoxyribose, C3-linker, and 2'-O-methyl-ribonucleoside; U2
is a naturally occurring nucleoside an immunostimulatory moiety
selected from the group consisting of 1',2'-dideoxyribose,
C3-linker, Spacer 18, 3'-deoxynucleoside, nucleoside
methylphosphonate, .beta.-L-deoxynucleosid- e, and
2'-O-propargyl-ribonucleoside; U3 is a naturally occurring
nucleoside an immunostimulatory moiety selected from the group
consisting of 1',2'-dideoxyribose, C3-linker, Spacer 9, Spacer 18,
nucleoside methylphosphonate, and 2'-5' linkage; D1 is a naturally
occurring nucleoside an immunostimulatory moiety selected from the
group consisting of 1',2'-dideoxyribose and nucleoside
methylphosphonate; D2 is a naturally occurring nucleoside an
immunostimulatory moiety selected from the group consisting of
1',2'-dideoxyribose, C3-linker, Spacer 9, Spacer 18,
2-aminobutyl-1,3-propanediol linker, nucleoside methylphosphonate,
and .beta.-L-deoxynucleoside; and D3 is a naturally occurring
nucleoside an immunostimulatory moiety selected from the group
consisting of 3'-deoxynucleoside, 2'-O-propargylribonucleoside; and
2'-5' linkage.
15. The immunostimulatory oligonucleotide compound of claim 13,
wherein U2 and U3 are both the same immunostimulatory moiety
selected from the group consisting of 1',2'-didoxyribose,
C3-linker, or .beta.-L-deoxynucleoside.
16. The immunostimulatory oligonucleotide compound of claim 13,
wherein U3 and U4 are both the same immunostimulatory moiety
selected from the group consisting of nucleoside methylphosphonate
and 2'-O-methoxyethylribonucle- oside.
17. The immunostimulatory oligonucleotide compound of claim 13,
wherein U5 and U6 are both the same immunostimulatory moiety
selected from the group consisting of 1',2'-dideoxyribose and
C3-linker.
18. The immunostimulatory oligonucleotide compound of claim 13,
wherein X1 and U3 are both 1',2'-dideoxyribose.
19. The immunostimulatory oligonucleotide compound of claim 13,
wherein D2 and D3 are both the same immunostimulatory moiety
selected from the group consisting of 1',2'-dideoxyribose and
.beta.-L-deoxynucleoside.
20. An immunostimulatory oligonucleotide compound, comprising: an
immunostimulatory dinucleotide of formula 5'-pyrimidine-purine-3',
wherein pyrimidine is a natural or non-natural pyrimidine
nucleoside and purine is a natural or non-natural purine
nucleoside; a 3'-3' linkage; and one or two accessible 5' ends;
provided that the oligonucleotide is not complementary to the gag
or tat gene of HIV-1.
21. The immunostimulatory oligonucleotide compound of claim 20,
which oligonucleotide comprises two accessible 5' ends.
22. The immunostimulatory oligonucleotide compound of claim 20,
wherein the immunostimulatory dinucleotide comprises a non-natural
pyrimidine nucleoside.
23. A method for modulating the immunostimulatory effect of an
immunostimulatory oligonucleotide compound, comprising introducing
into the immunostimulatory domain a dinucleotide analog that
includes a non-naturally occurring pyrimidine base.
24. A method for modulating the immunostimulatory effect of an
immunostimulatory oligonucleotide compound, comprising introducing
into the immunostimulatory domain and/or potentiation domain an
immunostimulatory moiety.
25. A method for modulating the immunostimulatory effect of an
immunostimulatory oligonucleotide compound, comprising introducing
into the oligonucleotide a 3'-3' linkage.
26. A method for generating an immune response in a patient, such
method comprising administering to the patient an oligonucleotide
analog immunostimulatory compound according to any one of claims 1,
2, 9, 12, and 20.
27. The method according to claim 26, wherein the oligonucleotide
analog immunostimulatory compound is administered in combination
with an antibiotic, antigen, allergen, vaccine, antibody, cytotoxic
agent, antisense oligonucleotide, gene therapy vector, DNA vaccine,
adjuvant, or combination thereof.
28. The method according to claim 26, wherein the immunostimulatory
oligonucleotide compound is conjugated to an antigen or
vaccine.
29. The method according to claim 28, wherein such conjugation is
to the 3'-end of the oligonucleotide compound.
30. A method for therapeutically treating a patient having disease
caused by a pathogen, such method comprising administering to the
patient an immunostimulatory oligonucleotide compound according to
any of claims 1, 2, 9, 12, and 20.
31. The method according to claim 30, wherein the pathogen is a
virus.
32. The method according to claim 30, wherein the pathogen is a
parasite.
33. The method according to claim 30, wherein the pathogen is a
bacterium.
34. A method for treating a cancer patient, such method comprising
administering to the patient an immunostimulatory oligonucleotide
compound according to any of claims 1, 2, 9, 12, and 20.
35. The method according to claim 34, wherein the immunostimulatory
oligonucleotide compound is administered in combination with a
chemotherapeutic compound.
36. A method for treating an autoimmune disorder, such method
comprising administering to the patient an oligonucleotide analog
immunostimulatory compound according to any of claims 1, 2, 9, 12,
and 20.
37. The method according to claim 36, wherein the autoimmune
disorder is autoimmune asthma.
38. A method for treating airway inflammation or allergy, such
method comprising administering to the patient an oligonucleotide
analog immunostimulatory compound according to any of claims 1, 2,
9, 12, and 20.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/712,898, filed on Nov. 15, 2000. This
application also claims priority from U.S. provisional patent
application serial Nos. 60/235,452 and 60/235,453, both filed on
Sep. 26, 2000. Each of the patent applications listed above is
hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to the therapeutic use of
oligonucleotides or oligonucleotide analogs as immunostimulatory
agents in immunotherapy applications.
[0004] 2. Summary of the Related Art
[0005] Oligonucleotides have become indispensable tools in modern
molecular biology, being used in a wide variety of techniques,
ranging from diagnostic probing methods to PCR to antisense
inhibition of gene expression and immunotherapy applications. This
widespread use of oligonucleotides has led to an increasing demand
for rapid, inexpensive and efficient methods for synthesizing
oligonucleotides.
[0006] The synthesis of oligonucleotides for antisense and
diagnostic applications can now be routinely accomplished. See
e.g., Methods in Molecular Biology, Vol 20: Protocols for
Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana
Press, 1993); Oligonucleotides and Analogues: A Practical Approach,
pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra.
Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense
Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca
Raton, 1993). Early synthetic approaches included phosphodiester
and phosphotriester chemistries. Khorana et al., J. Molec. Biol.
72: 209 (1972) discloses phosphodiester chemistry for
oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179
(1978), discloses phosphotriester chemistry for synthesis of
oligonucleotides and polynucleotides. These early approaches have
largely given way to the more efficient phosphoramidite and
H-phosphonate approaches to synthesis. Beaucage and Caruthers,
Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of
deoxynucleoside phosphoramidites in polynucleotide synthesis.
Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses
optimized synthesis of oligonucleotides by the H-phosphonate
approach.
[0007] Both of these modern approaches have been used to synthesize
oligonucleotides having a variety of modified internucleotide
linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542
(1987), teaches synthesis of oligonucleotide methylphosphonates
using phosphoramidite chemistry. Connolly et al., Biochemistry 23:
3443 (1984), discloses synthesis of oligonucleotide
phosphorothioates using phosphoramidite chemistry. Jager et al.,
Biochemistry 27: 7237 (1988), discloses synthesis of
oligonucleotide phosphoramidates using phosphoramidite chemistry.
Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988),
discloses synthesis of oligonucleotide phosphoramidates and
phosphorothioates using H-phosphonate chemistry.
[0008] More recently, several researchers have demonstrated the
validity of the use of oligonucleotides as immunostimulatory agents
in immunotherapy applications. The observation that phosphodiester
and phosphorothioate oligonucleotides can induce immune stimulation
has created interest in developing this side effect as a
therapeutic tool. These efforts have focused on phosphorothioate
oligonucleotides containing the dinucleotide CpG.
[0009] Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992)
teaches that phosphodiester oligonucleotides containing a
palindrome that includes a CpG dinucleotide can induce
interferon-alpha and gamma synthesis and enhance natural killer
activity. Krieg et al., Nature 371: 546-549 (1995) discloses that
phosphorothioate CpG-containing oligonucleotides are
immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129
(1996) discloses that such oligonucleotides activate human B
cells.
[0010] Pisetsky, D. S.; Rich C. F., Life Sci. 54: 101 (1994),
teaches that the immunostimulatory activity of CpG-oligos is
further enhanced by the presence of phosphorothioate (PS) backbone
on these oligos. Tokunaga, T.; Yamamoto, T.; Yamamoto, S. Jap. J.
Infect. Dis. 52: 1 (1999), teaches that immunostimulatory activity
of CpG-oligos is dependent on the position of CpG-motif and the
sequences flanking CpG-motif. The mechanism of activation of immune
stimulation by CpG-oligos has not been well understood. Yamamoto,
T.; Yamamoto, S.; Kataoka, T.; Tokunaga, T., Microbiol. Immunol.
38: 831 (1994), however, suggests that CpG-oligos trigger immune
cascade by binding to an intracellular receptor/protein, which is
not characterized yet.
[0011] Several researchers have found that this ultimately triggers
stress kinase pathways, activation of NF-.kappa.B and induction of
various cytokines such as IL-6, IL-12, .gamma.-IFN, and
TNF-.alpha.. (See e.g., Klinman, D. M.; Yi, A. K.; Beaucage, S. L.;
Conover, J.; Krieg, A. M., Proc. Natl. Acad. Sci. U.S. A. 93: 2879
(1996); Sparwasser, T.; Miethke, T.; Lipford, G. B.; Erdmann, A.;
Haecker, H.; Heeg, K.; Wagner, H., Eur. J. Immunol. 27: 1671
(1997); Lipford, G. B.; Sparwasser, T.; Bauer, M.; Zimmermann, S.;
Koch, E. S.; Heeg, K.; Wagner, H. Eur. J., Immunol. 27: 3420
(1997); Sparwasser, T.; Koch, E. S.; Vabulas, R. M.; Lipford, G.
B.; Heeg, K.; Ellart, J. W.; Wagner, H., Eur. J. Immunol. 28: 2045
(1998); and Zhao, Q.; Temsamani, J.; Zhou, R. Z.; Agrawal, S.
Antisense Nucleic Acid Drug Dev. 7: 495 (1997).)
[0012] The use of CpG-PS-oligos as antitumor, antiviral,
antibacterial and antiinflammatory agents and as adjuvants in
immunotherapy has been reported. (See e.g., Dunford, P. J.;
Mulqueen, M. J.; Agrawal, S. Antisense 97: Targeting the Molecular
Basis of Disease, (Nature Biotechnology) Conference abstract, 1997,
pp 40; Agrawal, S.; Kandimalla E. R. Mol. Med. Today 6:72 (2000);
Chu. R. S.; Targoni, O. S.; Krieg, A. M.; Lehmann, P. V.; Harding,
C. V. J. Exp. Med. 186: 1623 (1997); Zimmermann, S.; Egeter, 0.;
Hausmann, S.; Lipford, G. B.; Rocken, M.; Wagner, H.; Heeg, K. J.
Immunol. 160: 3627 (1998).) Moldoveanu et al., Vaccine 16: 1216-124
(1998) teaches that CpG-containing phosphorothioate
oligonucleotides enhance immune response against influenza virus.
McCluskie and Davis, J. Immunol. 161: 4463-4466 (1998) teaches that
CpG-containing oligonucleotides act as potent adjuvants, enhancing
immune response against hepatitis B surface antigen.
[0013] Zhao, Q.; Temsamani, J.; Idarola, P.; Jiang, Z.; Agrawal, S.
Biochem. Pharmacol. 51: 173 (1996), teaches that replacement of
deoxynucleosides in a CpG-motif with 2'-O-methylribonucleosides
suppresses immunostimulatory activity, suggesting that a rigid
C3'-endo conformation induced by 2'-O-methyl modification does not
allow proper recognition and/or interaction of CpG-motif with the
proteins involved in the immunostimulatory pathway. This reference
further teaches that substitution of a methyl group for an
unbridged oxygen on the phosphate group between C and G of a
CpG-motif suppresses immune stimulatory activity, suggesting that
negative charge on phosphate group is essential for protein
recognition and interaction.
[0014] Zhao, Q.; Yu, D.; Agrawal, S. Bioorg. Med. Chem. Lett. 9:
3453 (1999), teaches that substitution of one or two
2'-deoxynucleosides adjacent to CpG-motif with 2'- or
3'-O-methylribonucleosides on the 5'-side causes a decrease in
immunostimulatory activity, while the same substitutions have
insignificant effect when they were placed on the 3'-side of the
CpG-motif. However, Zhao, Q.; Yu, D.; Agrawal, S. Bioorg. Med.
Chem. Lett. 10: 1051 (2000), teaches that the substitution of a
deoxynucleoside two or three nucleosides away from the CpG-motif on
the 5'-side with one or two 2'-O-methoxyethyl- or 2'- or
3'-O-methylribonucleosides results in a significant increase in
immunostimulatory activity.
[0015] The precise structural requirements and specific functional
groups of CpG-motif necessary for the recognition of
protein/receptor factor that is responsible for immune stimulation
have not yet been studied in detail. There is, therefore, a need
for new immunostimulatory motifs which may provide improved
immunostimulatory activity.
BRIEF SUMMARY OF THE INVENTION
[0016] The invention provides methods for enhancing the immune
response caused by immunostimulatory oligonucleotide compounds. The
methods according to the invention enable increasing the
immunostimulatory effect for immunotherapy applications. Thus, the
invention further provides methods for making and using such
oligonucleotide compounds.
[0017] The present inventors have surprisingly discovered that
positional modification of immunostimulatory oligonucleotides
dramatically affects their immunostimulatory capabilities. In
particular, modifications in the immunostimulatory domain and/or
the potentiation domain enhance the immunostimulatory effect in a
reproducible and predictable manner.
[0018] In a first aspect, the invention provides immunostimulatory
oligonucleotide compounds comprising an immunostimulatory domain
and, optionally, one or more potentiation domains. In some
embodiments, the immunostimulatory domain comprises a dinucleotide
analog that includes a non-naturally occurring pyrimidine base. In
some embodiments, the immunostimulatory domain and/or the
potentiation domain include an immunostimulatory moiety at a
specified position, as described hereinbelow. In some embodiments,
the immunostimulatory oligonucleotide comprises a 3'-3' linkage. In
one embodiment, such 3'-3' linked oligonucleotides have two
accessible 5'-ends.
[0019] In a second aspect, the invention provides methods for
modulating the immunostimulatory effect of an immunostimulatory
oligonucleotide compound. In some embodiments, the method comprises
introducing into the immunostimulatory domain a dinucleotide analog
that includes a non-naturally occurring pyrimidine base. In some
embodiments, the method comprises introducing into the
immunostimulatory domain and/or potentiation domain an
immunostimulatory moiety at a specified position, as described
hereinbelow. In some embodiments, the method comprises introducing
into the oligonucleotide a 3'-3' linkage.
[0020] In a third aspect, the invention provides methods for
generating an immune response in a patient, such methods comprising
administering to the patient an immunostimulatory oligonucleotide
compound according to the invention.
[0021] In a fourth aspect, the invention provides methods for
therapeutically treating a patient having disease caused by a
pathogen, such methods comprising administering to the patient an
immunostimulatory oligonucleotide compound according to the
invention.
[0022] In a fifth aspect, the invention provides methods for
treating a cancer patient, such methods comprising administering to
the patient an immunostimulatory oligonucleotide compound according
to the invention.
[0023] In a sixth aspect, the invention provides methods for
treating autoimmune disorders, such as autoimmune asthma, such
methods comprising administering to the patient an oligonucleotide
analog immunostimulatory compound according to the invention.
Administration is carried out as described for the third aspect of
the invention.
[0024] In a seventh aspect, the invention provides methods for
treating airway inflammation or allergies, such methods comprising
administering to the patient an oligonucleotide analog
immunostimulatory compound according to the invention.
Administration is carried out as described for the third aspect of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows results of proliferation assays using
oligonucleotides having 1',2'-dideoxyribose substitutions at
various positions.
[0026] FIG. 2 shows results of spleen weight assays using
oligonucleotides having 1',2'-dideoxyribose substitutions at
various positions.
[0027] FIG. 3 shows results of proliferation assays using different
oligonucleotides having 1',2'-dideoxyribose substitutions at
various positions.
[0028] FIG. 4 shows results of spleen weight assays using different
oligonucleotides having 1',2'-dideoxyribose substitutions at
various positions.
[0029] FIG. 5 shows results of proliferation assays using
oligonucleotides having C3-linker substitutions at various
positions.
[0030] FIG. 6 shows results of spleen weight assays using
oligonucleotides having C3-linker substitutions at various
positions.
[0031] FIG. 7 shows results of proliferation assays using
oligonucleotides having Spacer 9 or Spacer 18 substitutions at
various positions.
[0032] FIG. 8 shows results of spleen weight assays using
oligonucleotides having Spacer 9 or Spacer 18 substitutions at
various positions.
[0033] FIG. 9 shows results of proliferation assays using
oligonucleotides having amino-linker substitutions at various
positions.
[0034] FIG. 10 shows results of spleen weight assays using
oligonucleotides having amino-linker substitutions at various
positions.
[0035] FIG. 11 shows results of proliferation assays using
oligonucleotides having 3'-deoxynucleoside substitutions at various
positions.
[0036] FIG. 12 shows results of spleen weight assays using
oligonucleotides having 3'-deoxynucleoside substitutions at various
positions.
[0037] FIG. 13 shows results of proliferation assays using
oligonucleotides having methylphosphonate substitutions at various
positions.
[0038] FIG. 14 shows results of spleen weight assays using
oligonucleotides having methylphosphonate substitutions at various
positions.
[0039] FIG. 15 shows results of proliferation assays using
oligonucleotides having 2'-O-methylribonucleoside or
2'-O-methoxyethyl substitutions at various positions.
[0040] FIG. 16 shows results of spleen weight assays using
oligonucleotides having 2'-O-methylribonucleoside or
2'-O-methoxyethyl substitutions at various positions.
[0041] FIG. 17 shows results of proliferation assays using
oligonucleotides having 5'-3', 5'-5', or 3'-3' linkage
substitutions at various positions.
[0042] FIG. 18 shows results of spleen weight assays using
oligonucleotides having .beta.-L-deoxynucleotide substitutions at
various positions.
[0043] FIG. 19 shows results of spleen weight assays using
oligonucleotides having 2'-O-propargyl substitutions at various
positions.
[0044] FIG. 20 shows results of spleen weight assays using
oligonucleotides having various substitutions at various
positions.
[0045] FIG. 21 shows results of spleen weight assays using
oligonucleotides having 7-deazaguanine substitution within the
immunostimulatory dinucleotide.
[0046] FIG. 22 shows results of proliferation assays using
oligonucleotides having 6-thioguanine substitution within the
immunostimulatory dinucleotide.
[0047] FIG. 23 shows results of spleen weight assays using
oligonucleotides having 5-hydroxycytosine or N4-ethylcytosine
substitution within the immunostimulatory dinucleotide.
[0048] FIG. 24 shows results of spleen weight assays using
oligonucleotides having 5-hydroxycytosine or N4-ethylcytosine
substitution within the immunostimulatory dinucleotide.
[0049] FIG. 25 shows results of proliferation assays using
oligonucleotides having arabinofuranosylcytosine (aracytidine;
Ara-C) substitution within the immunostimulatory dinucleotide.
[0050] FIG. 26 shows results of spleen weight assays using
oligonucleotides having 4-thiouracil substitution within the
immunostimulatory dinucleotide.
[0051] FIG. 27 shows the chemical structure of a CpG-motif, showing
functional groups on cytosine that serve as hydrogen bond acceptor
and hydrogen bond donor groups.
[0052] FIG. 28 shows the chemical structures of cytosine (1) and
cytosine analogs (2-7). In the nucleosides cytidine, deoxycytidine,
and related analogs, the substituent R is ribose or
2'-deoxyribose.
DETAILED DESCRIPTION
[0053] The invention relates to the therapeutic use of
oligonucleotides and oligonucleotide analogs as immunostimulatory
agents for immunotherapy applications. The patents and publications
cited herein reflect the level of knowledge in the field and are
hereby incorporated by reference in their entirety. In the event of
conflict between any teaching of any reference cited herein and the
present specification, the latter shall prevail, for purposes of
the invention.
[0054] The invention provides methods for enhancing the immune
response caused by immunostimulatory oligonucleotide compounds for
immunotherapy applications. Thus, the invention further provides
compounds having optimal levels of immunostimulatory effect for
immunotherapy and methods for making and using such oligonucleotide
compounds.
[0055] The present inventors have surprisingly discovered that
positional chemical modifications introduced in immunostimulatory
oligonucleotides dramatically affect their immunostimulatory
capabilities. In particular, modifications in the immunostimulatory
domain and/or the potentiation domain can enhance the
immunostimulatory effect in a reproducible manner for desired
applications.
[0056] In a first aspect, the invention provides immunostimulatory
oligonucleotide compounds comprising an immunostimulatory domain
and, optionally, one or more potentiation domains. In certain
preferred embodiments, the immunostimulatory domain comprises a
dinucleotide analog that includes a non-natural pyrimidine
nucleoside.
[0057] For purposes of all aspects of the invention, the term
"oligonucleotide" includes polymers of two or more
deoxyribonucleosides, or any modified nucleoside, including 2'- or
3'-substituted nucleosides, 2'- or 3'-O-substituted
ribonucleosides, deazanucleosides, or any combination thereof. Such
monomers may be coupled to each other by any of the numerous known
internucleoside linkages. In certain preferred embodiments, these
internucleoside linkages may be phosphodiester, phosphotriester,
phosphorothioate, phosphorodithioate, or phosphoramidate linkages,
including 3'-5', 2'-5', 3'-3', and 5'-5' linkages of any of the
foregoing, or combinations thereof. The term oligonucleotide also
encompasses such polymers having chemically modified bases or
sugars and/or having additional substituents, including without
limitation lipophilic groups, intercalating agents, diamines and
adamantane. The term oligonucleotide also encompasses peptide
nucleic acids (PNA), peptide nucleic acids with phosphate groups
(PHONA), locked nucleic acids (LNA), morpholinonucleic acids, and
oligonucleotides comprising non-pentose sugar (e.g. hexose) or
abasic sugar backbones or backbone sections, as well as
oligonucleotides that include backbone sections with non-sugar
linker or spacer groups, as further described hereinbelow.
[0058] For purposes of the invention the terms "2'-substituted" and
"3'-substituted" mean (respectively) substitution of the 2' (or 3')
position of the pentose moiety with a halogen (preferably Cl, Br,
or F), or an --O-lower alkyl group containing 1-6 saturated or
unsaturated carbon atoms, or with an --O-aryl or allyl group having
2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be
unsubstituted or may be substituted, e.g., with halo, hydroxy,
trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,
carbalkoxy, or amino groups; or such 2' substitution may be with a
hydroxy group (to produce a ribonucleoside) or an amino group, but
not with a 2' (or 3') H group.
[0059] For purposes of the invention, the term "immunostimulatory
oligonucleotide compound" means a compound comprising an
immunostimulatory dinucleotide, without which the compound would
not have an immunostimulatory effect. An "immunostimulatory
dinucleotide" is a dinucleotide having the formula
5'-pyrimidine-purine-3', wherein "pyrimidine" is a natural or
non-natural pyrimidine nucleoside and "purine" is a natural or
non-natural purine nucleoside. One such immunostimulatory
dinucleotide is CpG. The terms "CpG" and "CpG dinucleotide" refer
to the dinucleotide 5'-deoxycytidine-deoxyguanosine-3- ', wherein p
is an internucleotide linkage, preferably selected from the group
consisting of phosphodiester, phosphorothioate, and
phosphorodithioate.
[0060] For purposes of the invention, a "dinucleotide analog" is an
immunostimulatory dinucleotide as described above, wherein either
or both of the pyrimidine and purine nucleosides is a non-natural
nucleoside. A "non-natural" nucleoside is one that includes a
non-naturally occurring base and/or a non-naturally occurring sugar
moiety. For purposes of the invention, a base is considered to be
non-natural if it is not selected from the group consisting of
thymine, guanine, cytosine, adenine, and uracil. The terms "C*pG"
and "CpG*" refer to immunostimulatory dinucleotide analogs
comprising a cytidine analog (non-natural pyrimidine nucleoside) or
a guanosine analog (non-natural purine nucleoside),
respectively.
[0061] FIG. 27 shows the chemical structure of a CpG-motif, showing
the functional groups on cytosine that serve as hydrogen bond
acceptor and hydrogen bond donor groups. Cytosine has two hydrogen
bond acceptor groups at positions 2 (keto-oxygen) and 3 (nitrogen),
and a hydrogen bond donor group at the 4-position (amino group)
These groups can serve as potential recognizing and interacting
groups with receptors that are responsible for immune stimulation.
FIG. 28 shows cytosine analogs that are isostructural with natural
cytosine, including 5-methyl-deoxycytosine (2),
5-methyl-deoxyisocytosine (3), 5-hydroxy-deoxycytosine (4),
deoxyuridine (5), N4-ethyl-deoxycytosine (6), and deoxy-P-base
(7).
[0062] In one embodiment, therefore, the immunostimulatory
dinucleotide comprises a pyrimidine nucleoside of structure (I):
1
[0063] wherein D is a hydrogen bond donor, D' is selected from the
group consisting of hydrogen, hydrogen bond donor, hydrogen bond
acceptor, hydrophilic group, hydrophobic group, electron
withdrawing group and electron donating group, A is a hydrogen bond
acceptor, X is carbon or nitrogen, and S is a pentose or hexose
sugar ring linked to the pyrimidine base. In some embodiments, the
pyrimidine nucleoside is a non-natural pyrimidine nucleoside, i.e.,
the compound of structure (I) is not cytidine or deoxycytidine.
[0064] In some embodiments, the base moiety in (I) is a
non-naturally occurring pyrimidine base. Examples of preferred
non-naturally occurring pyrimidine bases include, without
limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine,
N4-alkylcytosine, preferably N4-ethylcytosine, and 4-thiouracil. In
some embodiments, the sugar moiety S in (I) is a non-naturally
occurring sugar moiety. For purposes of the present invention, a
"naturally occurring sugar moiety" is ribose or 2'-deoxyribose, and
a "non-naturally occurring sugar moiety" is any sugar other than
ribose or 2'-deoxyribose that can be used in the backbone for an
oligonucleotide. Arabinose and arabinose derivatives are examples
of a preferred non-naturally occurring sugar moieties.
[0065] Immunostimulatory domains according to the invention may
include immunostimulatory moieties on one or both sides of the
immunostimulatory natural dinucleotide or non-natural dinucleotide
analog. For example, an immunostimulatory domain could be depicted
as
5'-X1-X2-Y-Z-X3-X4-3'
[0066] wherein Y represents cytidine or a non-natural pyrimidine
nucleoside analog, Z represents guanosine or a non-natural purine
nucleoside analog, and each X independently represents a nucleoside
or an immunostimulatory moiety according to the invention. An
"immunostimulatory moiety" is a chemical structure at a particular
position within the immunostimulatory domain or the potentiation
domain that causes the immunostimulatory oligonucleotide to be more
immunostimulatory than it would be in the absence of the
immunostimulatory moiety.
[0067] Preferred immunostimulatory moieties include modifications
in the phosphate backbones including without limitation
methylphosphonates, methylphosphonothioates phosphotriesters,
phosphothiotriesters phosphorothioates, phosphorodithioates,
triester prodrugs, sulfones, sulfonamides, sulfamates, formacetal,
N-methylhydroxylamine, carbonate, carbamate, boranophosphonate,
phosphoramidates, especially primary amino-phosphoramidates, N3
phosphoramidates and N5 phosphoramidates, and stereospecific
linkages (e.g., (R)- or (S)-phosphorothioate, alkylphosphonate, or
phosphotriester linkages). Preferred immunostimulatory moieties
according to the invention further include nucleosides having sugar
modifications, including without limitation 2'-substituted pentose
sugars including without limitation 2'-O-methylribose,
2'-O-methoxyethylribose, 2'-O-propargylribose, and
2'-deoxy-2'-fluororibose; 3'-substituted pentose sugars, including
without limitation 3'-O-methylribose; 1',2'-dideoxyribose; hexose
sugars, including without limitation arabinose, 1'-methylarabinose,
3'-hydroxymethylarabinose, 4'-hydroxymethylarabinose, and
2'-substituted arabinose sugars; and alpha-anomers.
[0068] Preferred immunostimulatory moieties according to the
invention further include oligonucleotides having other
carbohydrate backbone modifications and replacements, including
peptide nucleic acids (PNA), peptide nucleic acids with phosphate
groups (PHONA), locked nucleic acids (LNA), morpholinonucleic
acids, and oligonucleotides having backbone sections with alkyl
linkers or amino linkers. The alkyl linker may be branched or
unbranched, substituted or unsubstituted, and chirally pure or a
racemic mixture. Most preferably, such alkyl linkers have from
about 2 to about 18 carbon atoms. In some preferred embodiments
such alkyl linkers have from about 3 to about 9 carbon atoms. Such
alkyl linkers include polyethyleneglycol linkers
[--O--CH2--CH2--].sub.n (n=2-9). In some preferred embodiments,
such alkyl linkers may include peptides or amino acids.
[0069] Preferred immunostimulatory moieties according to the
invention further include DNA isoforms, including without
limitation .beta.-L-deoxynucleosides and alpha-deoxynucleosides.
Preferred immunostimulatory moieties according to the invention
further include nucleosides having unnatural internucleoside
linkage positions, including without limitation 2'-5', 2'-2', 3'-3'
and 5'-5' linkages.
[0070] Preferred immunostimulatory moieties according to the
invention further include nucleosides having modified heterocyclic
bases, including without limitation 5-hydroxydeoxycytidine,
5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, preferably
N4-ethyldeoxycytidine, 4-thiouridine, 6-thiodeoxyguanosine,
7-deaza-guanosine, and deoxyribonucleosides of nitropyrrole,
C5-propynylpyrimidine, and diaminopurine, including without
limitation 2,6-diaminopurine.
[0071] By way of specific illustration and not by way of
limitation, for example, in the immunostimulatory domain described
earlier
5'-X1-X2-Y-Z-X3-X4-3'
[0072] a nucleoside methylphosphonate at position X3 or X4 is an
immunostimulatory moiety, a substituted or unsubstituted alkyl
linker at position X1 is an immunostimulatory moiety, and a
.beta.-L-deoxynucleosid- e at position X1 is an immunostimulatory
moiety. See Table 1 below for representative positions and
structures of immunostimulatory moieties within the
immunostimulatory domain.
1TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES X1 C3-alkyl
linker, 2-aminobutyl-1,3-propanediol linker (amino linker),
.beta.-L-deoxynucleoside X2 2-aminobutyl-1,3-propanediol linker X3
nucleoside methylphosphonate X4 nucleoside methylphosphonate,
2'-O-methyl-ribonucleoside
[0073] In some embodiments, the immunostimulatory oligonucleotide
further comprises a potentiation domain
[0074] A "potentiation domain" is a region of an immunostimulatory
oligonucleotide analog, other than the immunostimulatory domain,
that causes the oligonucleotide to be more immunostimulatory if it
contains the potentiation domain than the oligonucleotide would be
in the absence of the potentiation domain. The potentiation domain
can be upstream or downstream relative to the immunostimulatory
domain. The term "upstream" is used to refer to positions on the 5'
side of the immunostimulatory dinucleotide or dinucleotide analog
(Y-Z). The term "downstream" is used to refer to positions on the
3' side of Y-Z.
[0075] For example, an immunostimulatory oligonucleotide analog
could have the structure
5'-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-N--N--N-3'
[0076] wherein U9-U1 represents an upstream potentiation domain,
wherein each U independently represents the same or a different
nucleoside immunostimulatory moiety, N represents any nucleoside
and X1-X4, Y and Z are as before.
[0077] Alternatively, an immunostimulatory oligonucleotide analog
could have the structure
5'-N--N-X1-X2-Y-Z-X3-X4-D1-D2-D3-D4-D5-D6-D7-D8-3'
[0078] wherein D1-D8 represents a downstream potentiation domain,
wherein each D independently represents the same or a different
nucleoside or immunostimulatory moiety, and all other symbols are
as described above.
[0079] In these configurations, an immunostimulatory moiety at U6
would be six positions upstream from the immunostimulatory
dinucleotide or dinucleotide analog and an immunostimulatory moiety
at D4 would be four positions downstream from the immunostimulatory
dinucleotide or dinucleotide analog. The term "position" is used
rather than "nucleoside", because any of the U or D positions can
represent an immunostimulatory moiety which may or may not be a
nucleoside or nucleoside analog. Of course, oligonucleotide analogs
can be constructed having both upstream and downstream potentiation
domains.
[0080] Table 2 shows representative positions and structures of
immunostimulatory moieties within an immunostimulatory
oligonucleotide having an upstream potentiation domain. See FIG. 7
for definitions of Spacer 9 and Spacer 18 as referred to in Tables
2 and 3.
2TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY X2
2-aminobutyl-1,3-propanediol linker X1 C3-linker,
2-aminobutyl-1,3-propanediol linker, .beta.-L-deoxy- nucleoside U1
1',2'-dideoxyribose, C3-linker, 2'-O-methyl-ribonucleoside U2
1',2'-dideoxyribose, C3-linker, Spacer 18, 3'-deoxynucleoside,
nucleoside methylphosphonate, .beta.-L-deoxynucleoside, 2'-O-
propargyl-ribonucleoside U3 1',2'-dideoxyribose, C3-linker, Spacer
9, Spacer 18, nucleoside methylphosphonate, 2'-5' linkage U2 + U3
1',2'-dideoxyribose, C3-linker, ,.beta.-L-deoxynucleoside U3 + U4
nucleoside methylphosphonate, 2'-O-methoxyethyl- ribonucleoside U5
+ U6 1',2'-dideoxyribose, C3-linker X1 + U3 1',2'-dideoxyribose
[0081] Table 3 shows representative positions and structures of
immunostimulatory moieties within an immunostimulatory
oligonucleotide having a downstream potentiation domain.
3TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY X3 nucleoside
methylphosphonate X4 nucleoside methylphosphonate,
2'-O-methyl-ribonucleoside D1 1',2'-dideoxyribose, nucleoside
methylphosphonate D2 1',2'-dideoxyribose, C3-linker, Spacer 9,
Spacer 18, 2-aminobutyl-1,3-propanediol-linker, nucleoside
methylphosphonate, .beta.-L-deoxynucleoside D3 3'-deoxynucleoside,
2'-O-propargyl-ribonucleoside, 2'-5'-linkage D2 + D3
1',2'-dideoxyribose, .beta.-L-deoxynucleoside
[0082] In another embodiment of the invention, the oligonucleotide
according to the invention has one or two accessible 5' ends. The
present inventors have discovered that immunostimulatory moieties
in the region 5' to the immunostimulatory dinucleotide have a
greater impact on immunostimulatory activity than do similar
substitutions in the region 3' to the immunostimulatory
dinucleotide. This observation suggests that the 5'-flanking region
of CpG-PS-oligos plays an important role in immunostimulatory
activity. Moreover, the inventors have discovered that compounds
having two oligonucleotide units attached by way of a 3'-5' or
3'-3' linkage have greater immunostimulatory activity than do
compounds in which the two oligonucleotide units are attached by
way of a 5'-5' linkage. In some preferred embodiments, therefore,
the immunostimulatory oligonucleotide according to the invention
comprises a 3'-3' linkage. In some such embodiments, the
oligonucleotides have one or two accessible 5' ends.
[0083] In a second aspect, the invention provides methods for
modulating the immunostimulatory effect of an immunostimulatory
oligonucleotide. In some embodiments, the method comprises
introducing into the immunostimulatory domain a dinucleotide analog
that includes a non-naturally occurring pyrimidine base, as
described above for the first aspect of the invention. In some
embodiments, the method comprises introducing into the
immunostimulatory domain and/or potentiation domain an
immunostimulatory moiety at a specified position, as described
above. In some embodiments, the method comprises introducing into
the oligonucleotide a 3'-3' linkage.
[0084] For purposes of the invention, "introducing an
immunostimulatory moiety" at a specified position simply means
synthesizing an oligonucleotide that has an immunostimulatory
moiety at the specified position. For example, "introducing an
immunostimulatory moiety into position U6" simply means
synthesizing an oligonucleotide that has an immunostimulatory
moiety at such a position, with reference to, e.g., the following
structure:
5'-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-D1-D2-D3-3'.
[0085] Preferably, the methods according to this aspect of the
invention include introducing an immunostimulatory moiety at a
position in the immunostimulatory domain or in an upstream or
downstream potentiation domain according to the preferred
substitution patterns described in Tables 1-3.
[0086] The methods according to this aspect of the invention can be
conveniently carried out using any of the well-known synthesis
techniques by simply using an appropriate immunomodulatory moiety
monomer synthon in the synthesis process in an appropriate cycle to
obtain the desired position. Preferred monomers include
phosphoramidites, phosphotriesters and H-phosphonates. PS-oligos
are readily synthesized, e.g., using
.beta.-cyanoethylphosphoramidite chemistry on CPG solid support
using appropriate phosphoramidites, deprotected as required,
purified by C.sub.18 reverse phase HPLC, dialyzed against distilled
water and lyophilized. The purity of each PS-oligo is readily
determined by CGE and the molecular weight can be confirmed by
MALDI-TOF mass spectral analysis.
[0087] In a third aspect, the invention provides methods for
generating an immune response in a patient, such methods comprising
administering to the patient an oligonucleotide analog
immunostimulatory compound according to the invention.
[0088] In the methods according to this aspect of the invention,
preferably, administration of compounds is parenteral, oral,
sublingual, transdermal, topical, intranasal, intratracheal,
intravaginal, or intrarectal. Administration of the therapeutic
compositions can be carried out using known procedures at dosages
and for periods of time effective to reduce symptoms or surrogate
markers of the disease. When administered systemically, the
therapeutic composition is preferably administered at a sufficient
dosage to attain a blood level of oligonucleotide from about 0.001
micromolar to about 10 micromolar. For localized administration,
much lower concentrations than this may be effective, and much
higher concentrations may be tolerated. Preferably, a total dosage
of oligonucleotide will range from about 0.1 mg oligonucleotide per
patient per day to about 40 mg oligonucleotide per kg body weight
per day. It may be desirable to administer simultaneously, or
sequentially a therapeutically effective amount of one or more of
the therapeutic compositions of the invention to an individual as a
single treatment episode. In some instances, dosages below the
above-defined ranges may still provide efficacy. In a preferred
embodiment, after the composition of matter is administered, one or
more measurement is taken of biological effects selected from the
group consisting of complement activation, mitogenesis and
inhibition of thrombin clot formation.
[0089] In certain preferred embodiments, compounds according to the
invention are administered in combination with antibiotics,
antigens, allergens, vaccines, antibodies, cytotoxic agents,
antisense oligonucleotides, gene therapy vectors, DNA vaccines
and/or adjuvants to enhance the specificity or magnitude of the
immune response. Either the compound or the vaccine, or both may
optionally be linked to an immunogenic protein, such as keyhole
limpet hemocyanin, cholera toxin B subunit, or any other
immunogenic carrier protein. Any of a plethora of adjuvants may be
used, including, without limitation, Freund's complete adjuvant,
monophosphoryl lipid A (MPL), saponins, including QS-21, alum, and
combinations thereof. Certain preferred embodiments of the methods
according to the invention induce cytokines by administration of
immunostimulatory oligonucleotide compounds. In certain embodiments
the immunostimulatory oligonucleotide compounds are conjugated to
an antigen, hapten, or vaccine. As discussed above, the present
inventors have discovered that an accessible 5' end is important to
the activity of certain immunostimulatory oligonucleotide
compounds. Accordingly, for optimum immunostimulatory activity, the
oligonucleotide preferably is conjugated to an antigen or vaccine
by means of the 3'-end of oligonucleotide compound.
[0090] For purposes of this aspect "in combination with" means in
the course of treating the same disease in the same patient, and
includes administering the oligonucleotide and/or the vaccine
and/or the adjuvant in any order, including simultaneous
administration, as well as temporally spaced order of up to several
days apart. Such combination treatment may also include more than a
single administration of the oligonucleotide, and/or independently
the vaccine, and/or independently the adjuvant. The administration
of the oligonucleotide and/or vaccine and/or adjuvant may be by the
same or different routes.
[0091] The method according to this aspect of the invention is
useful for model studies of the immune system, and is further
useful for the therapeutic treatment of human or animal
disease.
[0092] In a fourth aspect, the invention provides methods for
therapeutically treating a patient having disease caused by a
pathogen, such methods comprising administering to the patient an
oligonucleotide analog immunostimulatory compound according to the
invention. Administration is carried out as described for the third
aspect of the invention.
[0093] In a fifth aspect, the invention provides methods for
treating a cancer patient, such methods comprising administering to
the patient an oligonucleotide analog immunostimulatory compound
according to the invention. Administration is carried out as
described for the third aspect of the invention.
[0094] In a sixth aspect, the invention provides methods for
treating autoimmune disorders, such as autoimmune asthma, such
methods comprising administering to the patient an oligonucleotide
analog immunostimulatory compound according to the invention.
Administration is carried out as described for the third aspect of
the invention.
[0095] In a seventh aspect, the invention provides methods for
treating airway inflammation or allergies, such methods comprising
administering to the patient an oligonucleotide analog
immunostimulatory compound according to the invention.
Administration is carried out as described for the third aspect of
the invention.
[0096] The following examples are intended to further illustrate
certain preferred embodiments of the invention, and are not
intended to limit the scope of the invention.
EXAMPLES
Example 1
Synthesis of Oligonucleotides Containing Immunomodulatory
Moieties
[0097] Oligonucleotides were synthesized on a 1 micromolar scale
using an automated DNA synthesizer (Expedite 8909, PerSeptive
Biosystems, Foster City, Calif.). Standard deoxynucleoside
phosphoramidites are obtained from PerSeptive Biosystems.
1',2'-dideoxyribose phosphoramidite, propyl-1-phosphoramidite,
2'-deoxy-5-nitroindole-ribofuranosyl phosphoramidite,
2'-deoxy-uridine phosphoramidite, 2'-deoxy-P phosphoramidite,
2'-deoxy-2-aminopurine phosphoramidite, 2'-deoxy-nebularine
phosphoramidite, 2'-deoxy-7-deazaguanosine phosphoramidite,
2'-deoxy-4-thiouridine phosphoramidite, 2'-deoxy-isoguanosine
phosphoramidite, 2'-deoxy-5-methylisocytosine phosphoramidite,
2'-deoxy-4-thiothymidine phosphoramidite,
2'-deoxy-K-phosphoramidite, 2'-deoxy-2-aminoadenosine
phosphoramidite, 2'-deoxy-N4-ethyl-cytosine phosphoramidite,
2'-deoxy-6-thioguanosine phosphoramidite,
2'-deoxy-7-deaza-xanthosine phosphoramidite,
2'-deoxy-8-bromoguanosine phosphoramidite, 2'-deoxy-8-oxoguanosine
phosphoramidite, 2'-deoxy-5-hydroxycytosine phosphoramidite,
arabino-cytosine phosphoramidite and 2'-deoxy-5-propynecytosine
phosphoramidite were obtained from Glen Research (Sterling, Va.).
2'-Deoxy-inosine phosphoramidite were obtained from ChemGenes
(Ashland, Mass.).
[0098] Normal coupling cycles or a coupling cycle recommended by
the phosphoramidite manufacturer were used for all
phosphoramidites. Beaucage reagent was used as an oxidant to obtain
phosphorothioate modification. After synthesis, oligonucleotides
were deprotected by incubating CPG-bound oligonucleotide with
concentrated ammonium hydroxide solution for 1.5-2 hours at room
temperature and then incubating the ammonium hydroxide supernatant
for 12 hours at 55 degrees C or as recommended by phosphoramidite
manufacturer. The ammonium hydroxide solution was evaporated to
dryness in a speed-vac and 5'-DMTr-oligonucleotides were purified
by HPLC on a C18 reverse-phase matrix using a solvent system of 0.1
M ammonium acetate and 1:5 ratio 0.1 M ammonium acetate in
acetonitrile. Then the oligonucleotides were treated with 80%
acetic acid to remove the DMTr group, converted to sodium form and
desalted by dialysis against double distilled water.
Oligonucleotides were filtered through 0.4 .mu. filters,
lyophilized and redissolved in double distilled water.
Characterization was achieved by denaturing PAGE and MALDI-TOF mass
spectrometry.
Example 2
Synthesis of CpG-PS-Oligos Containing Cytosine Analogs
[0099] Following the procedures outlined in Example 1, the
following oligonucleotides were synthesized:
4 Oligo # Sequence (5'--->3') and Modification.sup.a 1
d(CTATCTGACGTTCTCTGT) 2 d(CTATCTGAC*GTTCTCTGT) 3
d(CTATCTGACC*TTCTCTGT) 4 d(CTATCTGAC*GTTCTCTGT) 5
d(CTATCTGACC*TTCTCTGT) .sup.aCpG-motif is shown in bold. C*
represents 5-hydroxycytosine (oligos 2 and 3) or N4-ethylcytosine
(oligos 4 and 5).
[0100] The oligonucleotides were characterized by CGE and MALDI-TOF
mass spectrometry (Brucker Proflex III MALDI-TOF mass spectrometer
with 337 nm N2 laser). Molecular weights observed and calculated
(shown in parentheses) for each oligonucleotide are as follows:
Oligo 1, 5704 (5704.8); Oligo 2, 5720 (5720.8); Oligo 3, 5681
(5680.7); Oligo 4, 5733 (5733); Oligo 5, 5694 (5693).
Example 3
Analysis of Spleen Weights in Treated Mice
[0101] Female BALB/c mice (4-5 weeks, 19-21 g, Charles River,
Wilmington, Mass.) were used in the study. The animals were fed
with commercial diet and water ad lib. The animals were injected
intraperitoneally with 5 or 10 mg/kg dose of immunostimulatory
oligonucleotide compound dissolved in sterile PBS. One group of
mice received PBS alone to serve as a control (PBS). Four animals
were used for each immunostimulatory oligonucleotide compound. Mice
were sacrificed 72 h later, spleens were harvested and weighed.
Example 4
Analysis of Immunostimulatory Oligonucleotide Compounds in Mouse
Lymphocyte Proliferation Assay
[0102] Spleens from CD-1, BALB/c, C57BL/6 mouse (4-8 weeks) were
used as source of lymphocytes. Single cell suspensions were
prepared by gently mincing with the frosted ends of glass slides.
Cells were then cultured in RPMI complete medium [RPMI medium
supplemented with 10% fetal bovine serum (FBS) (heat-inactivated at
56.degree. C. for 30 min), 50 .mu.uM 2-mercaptoethanol, 100 U/mL
penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine]. The cells
were then plated in 96-well dishes at a density of 10.sup.6
cells/mL in a final volume of 100 .mu.L. Immunostimulatory
oligonucleotide compounds or LPS (lipopolysaccharide) were added to
the cell culture in 10 .mu.L of TE buffer (10 mM Tris-HCl, pH 7.5,
1 mM EDTA). The cells were then set to culture at 37.degree. C.
After 44 h, 1 .mu.Ci .sup.3H-uridine (Amersham, Arlington Heights,
Ill.) was added to the culture in 20 .mu.L of RPMI medium, and the
cells were pulse-labeled for another 4 h. The cells were harvested
by automatic cell harvester (Skatron, Sterling, Va.), and the
filters were counted by a scintillation counter. The experiments
were performed in triplicate.
Example 5
Lymphocyte Proliferatory Activity of CpG-PS-Oligos Containing
Cytosine Analogs
[0103] The immunostimulatory activity of CpG-PS-oligos 1-5 (Example
4) was studied using a BALB/c mouse lymphocyte proliferation assay.
In brief, mouse spleen cells were cultured and incubated with
CpG-PS-oligos at 0.1, 0.3, 1.0 and 3.0 .mu.g/mL concentration for
48 hr and cell proliferation was measured by .sup.3H-uridine
incorporation.
[0104] FIG. 23 shows the dose-dependent cell proliferatory activity
of oligos 1-5 in mouse lymphocyte cultures. At a dose of 3.0
.mu.g/mL, oligo 1, with natural cytidine, showed a proliferation
index of 29.5.+-.2.1. Oligo 2, in which the cytosine base of the
deoxycytidine of the CpG-motif is replaced with a
5-hydroxycytosine, also showed dose-dependent lymphocyte
proliferation. A proliferation index of 23.7.+-.2.9 at 3.0 .mu.g/mL
dose was observed for oligo 2. PS-Oligo 4, which contained
N4-ethyl-cytosine in place of the cytosine base in the CpG-motif,
also showed dose-dependent cell-proliferation activity. The
proliferation index of 18.7.+-.1.6 observed for oligo 4 at a dose
of 3 .mu.g/mL suggests that the presence of a bulky hydrophobic
substitution on the 4-amino group of cytosine in a CpG-motif
slightly impedes immunostimulatory activity.
[0105] Oligo 3, in which 5-hydroxy-deoxycytidine was placed in the
deoxyguanosine position instead of the deoxycytidine position of
the CpG-motif, showed a proliferation index that was similar to
that observed for media control (FIG. 23). Similarly, the control
Oligo 5 in which deoxyguanosine in the CpG-motif was substituted
with N4-ethyldeoxycytidine, showed cell proliferation similar to
that of media control.
[0106] Other oligos, in which cytosine base in the CpG-motif was
replaced with 5-methyl-deoxycytosine (2; see FIG. 28),
5-methyl-deoxyisocytosine (3), deoxyuridine (5), or deoxy-P-base
(7) showed no or insignificant cell proliferatory activity in the
same assay system. These results suggest that (i) cell
proliferatory activity is maintained when the cytosine base of the
CpG motif is replaced with 5-hydroxycytosine or N4-ethylcytosine
(Oligos 2 and 4, respectively), but (ii) substitution of the
guanine base with these cytosine analogs results in a loss of cell
proliferatory activity.
Example 6
Splenomegaly in Mice Induced by CpG-PS-Oligos containing Cytosine
Analogs
[0107] To confirm the in vitro effects of CpG-PS-oligos, Oligos 1,
2, and 4 (from Example 4) were injected intraperitoneally (ip) to
BALB/c mice at a dose of 10 mg/kg and the change in spleen weight
was measured as an indicator of the level of immunostimulatory
activity of each PS-oligo. The change in spleen weight as a result
of treatment with CpG-PS-oligos is presented in FIG. 24. Female
BALB/c mice (4-6 weeks, 19-21 gm) were divided in to different
groups with four mice in each group. Oligonucleotides were
dissolved in sterile PBS and administered intraperitoneally to mice
at a dose of 10 mg/kg. After 72 hr, mice were sacrificed and
spleens were harvested and weighed. Each circle represents the
spleen weight of an individual mouse and the + represents the mean
spleen weight for each group.
[0108] Oligo 1, which has natural deoxycytidine in the CpG-motif,
showed about 45% increase in spleen weight at a dose of 10 mg/kg,
compared with the control group of mice that received PBS. Oligo 2,
which has a 5-hydroxycytosine in place of the cytosine base in the
CpG-motif, showed about 35% increase in spleen weight at the same
dose. Oligo 4, which has N4-ethylcytosine in place of the cytosine
base in the CpG-motif, showed about 34% increase in spleen weight
at the same dose compared to the control group. These data confirm
the results observed in lymphocyte proliferation assays for these
oligos containing modified cytidine analogs in place of
deoxycytidine in the CpG-motif.
Example 7
Structure-Activity Relationships of C*pG-PS-Oligos
[0109] The presence of a methyl group at the 5-position of cytosine
(5-methyl-deoxycytosine, 2 (FIG. 28)) in a CpG-motif completely
abolishes CpG related immunostimulatory effects of CpG-PS-oligos.
Based on the results observed in in vitro and in vivo experiments
we have constructed structure-activity relationships for the
PS-oligos containing cytosine analogs.
[0110] The replacement of the cytosine base (1) in the CpG-motif
with 5-methyl-isocytosine (3) resulted in complete loss of
immunostimulatory activity, as is the case with 5-methylcytosine
(2), which could be as a result of switching the keto and amino
groups at the 2 and 4-positions, respectively, and/or placing a
hydrophobic methyl group at the 5-position of cytosine.
[0111] Oligo 2, containing a hydrophilic hydroxy substitution at
the 5-position of the cytosine in the CpG-motif, showed
immunostimulatory activity similar to that of oligo 1, which
contains the natural cytosine base. This observation suggests that
bulky hydrophilic groups are better tolerated than are hydrophobic
groups at the 5-position of cytosine for immunostimulatory activity
of CpG-PS-oligos. Perhaps the binding pocket for the CpG-oligos on
receptor is hydrophilic in nature and can not accommodate a
hydrophobic group at the 5-position of cytosine.
[0112] When the cytosine base in the CpG-motif is replaced with
uracil (5 (see FIG. 28)), in which keto groups are present at both
the 2 and 4-positions, no immunostimulatory activity was observed,
suggesting that a hydrogen bond donor amino group at the 4-position
of cytosine is critical for immunostimulatory activity. When a
large hydrophobic ethyl group is placed on 4-amino group of
cytosine in a CpG-motif, reduced lymphocyte proliferation and a
slightly reduced increase in spleen weight in mice were observed,
suggesting that a bulky ethyl group at this position does not
interfere with binding of the CpG-PS-oligo to the receptor factors
responsible for immunostimulatory activity. In spite of the ethyl
substitution, the 4-amino group of N4-ethylcytosine (6) can
participate in hydrogen bond formation with an acceptor. The
modified pyrimidine base dP, in which the nitrogen group located at
the 4-position involved in ring structure formation with the
5-position, and which does not have a hydrogen bond donor amino
group at the 4-position, had no mouse lymphocyte proliferation
activity in cultures, suggesting that the 4-amino group of cytosine
in a CpG-motif is critical for immunostimulatory activity.
[0113] In conclusion, the results presented here show that the
functional groups at 2, 3, and 4 positions of the cytosine are
important for CpG-related immunostimulatory activity. A hydrophobic
substitution at the 5-position of cytosine completely suppresses
immunostimulatory activity of a CpG-oligo, while a hydrophilic
group at this position is tolerated well. In addition, the
immunostimulatory activity of CpG-PS-oligos containing
5-hydroxycytosine or N4-ethylcytosine in place of cytosine in the
CpG-motif can be modulated significantly by incorporating
appropriate chemical modifications in the 5'-flanking sequence,
suggesting that these cytosine analogs in a CpG-motif are
recognized as part of an immunostimulatory motif.
Example 8
Synthesis of End-Blocked CpG-PS Oligonucleotides
[0114] The CpG-PS-oligos shown in FIG. 17 were synthesized using an
automated synthesizer and phosphoramidite approach. Oligo 1
(16-mer) was synthesized using
nucleoside-5'-.beta.-cyanoethylphosphoramidites. Oligo 2, a 32-mer,
was synthesized using nucleoside-3'-.beta.-cyanoethylphospho-
ramidites and controlled pore glass support (CPG-solid support)
with a 3'-linked nucleoside in which 16-mer sequence of Oligo 1 was
repeated twice; therefore, Oligo 2 had two 16-mers (Oligo 1) linked
by a normal 3'-5'-linkage. Oligo 3, a 32-mer, was synthesized with
two 16-mers (Oligo 1) linked by a 5'-5'-linkage, so Oligo 3 had two
3'-ends and no 5'-end. Synthesis of Oligo 3 was carried out in two
steps: the first 16-mer was synthesized using
nucleoside-3'-P-cyano-ethylphosphoramidites and solid support with
a 3'-linked nucleoside, and then synthesis of the second 16-mer
segment was continued using nucleoside-5'-.beta.-cyano-ethylphosph-
oramidites. Oligo 4, a 32-mer, comprised two 16-mers (Oligo 1)
linked by a 3'-3'-linkage, so Oligo 4 had two 5'-ends and no
3'-end. Synthesis of Oligo 4 was carried out in two steps: the
first 16-mer was synthesized using
nucleoside-5'-.beta.-cyanoethylphosphoramidites and solid support
with a 5'-linked nucleoside, and the synthesis of the second 16-mer
segment was continued using
nucleoside-3'-.beta.-cyanoethylphosphoramidit- es. Synthesis of
Oligos 5-8 was carried out by using the same
nucleoside-.beta.-cyanoethylphosphoramidites as for Oligos 1-4,
respectively. At the end of the synthesis, Oligos 1-8 were
deprotected with concentrated ammonia solution, purified by
reversed phase HPLC, detritylated, desalted and dialyzed. The
purity of each PS-oligo was checked by CGE and the molecular weight
was confirmed by MALDI-TOF mass spectral analysis (Table 1). The
sequence integrity and directionality of 5'-CpG motif in Oligos 1-8
were confirmed by recording melting temperatures (Tms) of the
duplexes with their respective DNA complementary strands
(5'-AAGGTCGAGCGTTCTC-3' for Oligos 1-4, and
5'-ATGGCGCACGCTGGGAGA-3' for Oligos 5-8). The T.sub.ms of these
duplexes were 53.9.+-.0.9.degree. C. (Oligos 1-4), 61.8.degree. C.
(Oligo 5), and 58.8.+-.0.6.degree. C. (Oligos 6-8) (note that Oligo
5 was a 18-mer and Oligos 6-8 were 32-mers but not 36-mers).
Example 9
Mouse Spleen Lymphocyte Proliferatory Activity of End-Blocked
CpG-PS Oligonucleotides
[0115] Immunostimulatory activity of the end-blocked CpG-PS-oligos
of Example 8 was studied initially in a lymphocyte proliferation
assay. Typically, mouse (Balb-C) spleen lymphocytes were cultured
with CpG-PS-oligos at concentrations of 0.1, 1.0, and 10.0 .mu.g/ml
for 48 h and cell proliferation was determined by .sup.3H-uridine
incorporation, as described in Example 3. Results are shown in FIG.
17
[0116] Oligo 1 induced a dose-dependent effect on cell
proliferation; at a concentration of 10 .mu.g/ml (.about.2.0
.mu.M), the proliferation index was 5.0.+-.0.32. Oligo 2, which
consisted of two units of Oligo 1 linked by a 3'-5'-linkage, had a
proliferation index of 5.8.+-.0.28 at the same dose (.about.1.0
.mu.M). Oligo 3, which consisted of two units of Oligo 1 linked by
a 5'-5'-linkage, had a proliferation index of 2.0.+-.0.26,
reflecting a significantly lower immunostimulatory activity than
observed with Oligos 1 and 2. Oligo 4, which consisted of two units
of Oligo 1 linked by a 3'-3'-linkage, had a proliferation index of
7.2.+-.0.5, reflecting a greater immunostimulatory activity than
observed with Oligos 1 and 2.
[0117] Similar results were obtained with Oligos 5-8. Oligo 5 had a
proliferation index of 3.9.+-.0.12. Oligos 6-8, in which two units
of Oligo 5 are linked by a 3'-5'-linkage (Oligo 6), 5'-5'-linkage
(Oligo 7), and 3'-3'-linkage (Oligo 8) had proliferation indices of
4.9.+-.0.2, 1.74.+-.0.21, and 7.7.+-.0.82, respectively. Comparison
of the results obtained with Oligos 6-8 show that Oligos 6 and 8,
in which two Oligo 5 sequences were linked by a 3'-5'-linkage or a
3'-3'-linkage had greater immunostimulatory activity, while Oligo
7, in which two Oligo 5 were linked by a 5'-5'-linkage had
significant less immunostimulatory activity, than did Oligo 5.
[0118] Based on lymphocyte proliferation results of Oligos 1-8, it
is clear that when oligos are linked through their 5'-ends, there
is a significant loss of immunostimulatory activity, while if they
are linked through their 3'-ends, there is an increase in
immunostimulatory activity. It is important to note that
3'-3'-linked oligos have shown substantially greater stability
towards degradation by exonucleases than the oligos that contained
a free 3'-end, which could also result in increased
immunostimulatory activity. The lower immunostimulatory activity of
Oligos 3 and 7, in which the 5'-end of oligos is blocked, suggests
that accessibility to 5'-end of oligo is essential for
immunostimulatory activity of CpG-PS-oligos.
Example 10
Splenomegaly in Mice Induced by End-Blocked CpG-PS
Oligonucleotides
[0119] To confirm the immunostimulatory activity of Oligos 1-8
(Example 8) in vivo, a dose of 5 mg/kg of oligonucleotides was
injected intraperitoneally to Balb-C mice. The mice were sacrificed
72 hours post-administration, spleens were removed, blotted to
dryness, and weighed. Change in spleen weight in treated and
untreated mice was used as a parameter for immunostimulatory
activity.
[0120] Administration 5 mg/kg dose of Oligo 1 caused about 40%
increase in spleen weight compared with the control mice that
received PBS. Administration of Oligos 2 and 4 also caused about
50% increase in spleen weight. Administration of Oligo 3 caused no
difference in spleen weight compared with control mice. These
results further support the observation that Oligo 3, in which
5'-end was blocked, had significantly less immunostimulatory
activity compared to oligos that had accessible 5'-end. These
results were also confirmed with the administration of Oligos 5-8.
Administration of Oligos 5, 6, and 8 caused about 40-50% increase
in spleen weight, whereas no change in spleen weight was observed
following the administration of Oligo 7.
[0121] The above results suggest that the immunostimulatory
activity of PS-oligos containing a CpG motif is significantly
minimized if the 5'-end of the oligo is not accessible. This loss
in immunostimulatory activity of Oligos 3 and 7 cannot be explained
based on nuclease stability, as both oligos have two 3'-ends and
are not more susceptible to 3'-exonuclease degradation than are
Oligos 1, 2, 5, and 6, which have one 3'-end. PS-Oligos 4 and 8,
which have their 3'-ends blocked and are very stable to degradation
by exonucleases, showed similar immunostimulatory activity. Oligos
4 and 8 may show sustained immunostimulatory activity due to their
increased in vivo stability, which is not evident in the present
study as mice were sacrificed at only 72 hours after
administration. Studies are in progress in which mice will be
sacrificed at times later than 72 hours after administration.
[0122] The results described here are intriguing and suggest that
the 5'-end of CpG-PS-oligos is critical for immunostimulatory
activity. As discussed here, we have shown that substitution of
deoxynucleosides in 5'-flanking regions by modified 2'- or
3'-substituted ribonucleosides resulted in increased
immunostimulatory activity. In addition, substitution of
deoxynucleosides immediately upstream (5'-end) to the CpG motif
caused a significant suppression and substitution of
deoxynucleosides immediately downstream (3'-end) to the CpG motif
had no effect on immunostimulatory activity. Taken together, these
results suggest that the enzyme/receptor responsible for the
immunestimulation recognizes the CpG motif in oligos from the
5'-end and requires accessibility to the 5'-end.
[0123] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made
without departing from the true scope of the invention and appended
claims.
* * * * *